Linkage maps consisting primarily of molecular markers have been constructed in pea and apple. Different approaches have been taken to generate these maps. For pea, F2 and recombinant inbred populations have been used to study segregating loci, and a critical factor has been the selection of sufficiently divergent inbred parents for the crosses. In contrast, a double pseudotestcross format has been used in apple, and virutally every variety possesses sufficient heterozygosity to permit the development of a map by examination of the F1. Markers have been identified for many genes in each crop.
Fifty-four apple (Malus domestica Borkh.) cultivars were characterized electrophoretically using 6 isozyme systems. Intracultivar variation in isozyme phenotype was not observed, whereas intercultivar polymorphism was sufficient to permit reliable and unambiguous identification of nearly every cultivar. The most useful isozyme systems for distinguishing among the cultivars were 6-phosphogluconate dehydrogenase and aspartate aminotransferase. Sports could not be distinguished from the original cultivar. The genetic basic of several polymorphisms was known, enabling the comparison of the isozyme genotype observed in a hybrid with that predicted on the basis of parental genotypes. The 6-phosphogluconate dehydrogenase genotype of ‘Spartan’ indicated that ‘Yellow Newtown’ may not have been the paternal parent.
The ribosomal genes of the two crab apple (Malus) genotypes White Angel' and `Robusta 5' were characterized to determine the extent of between- and within-genotype heterogeneity. Initial investigations with a cloned sequence of soybean rDNA failed to detect some Malus intergenic spacer region fragments. An alternative probing method that used electrophoretically purified Malus rDNA was developed. Double-digests of total genomic DNA with combinations of 13 restriction endonucleases identified the positions of 35 restriction sites. Restriction site polymorphism was observed both between and within the crab apple genotypes. Ribosomal DNA from White Angel' was cloned in phage and plasmid vectors and mapped with 11 enzymes. The region of the spacer causing length heterogeneity was identified. These clones should be useful as genetic markers and for examining population dynamics and systematic of Malus and closely related taxa.
The polymorphism in nine enzyme systems in apple (Malus domestica Borkh.) was analyzed using horizontal starch gel electrophoresis. The systems studied included aspartate aminotransferase, diaphorase, glucosephosphate isomerase, isocitrate dehydrogenase, phosphoglucomutase, and triosephosphate isomerase. The products of at least 27 loci could be distinguished in these systems, 19 of which displayed polymorphism. Joint segregation analysis in populations derived from crosses between highly heterozygous cultivars revealed four multilocus linkage groups: Aat-c–Idh-1, Dia-2–Mdh-4, Gpi-c2-Aat–p, and (Dia-5, Pgm-p1)–(Mdh-2, Tpi-c2). Although several of the populations investigated had been prescreened for resistance to apple scab, cedar-apple rust, or fire blight, no correlation could be established between the inheritance of an allozyme and a resistant phenotype. The high frequency of duplicate loci encountered is in accordance with the postulated tetraploid nature of the genome.
Two triploid apple (Malus domestica Borkh.) cultivars, ‘Spigold’ (‘Red Spy’ x ‘Golden Delicious’) and ‘Jonagold’ (‘Golden Delicious’ x ‘Jonathan’), and their parental cultivars were analyzed for a number of isozyme systems by starch gel electrophoresis. The banding pattern and intensity of one isozyme system, 6-phosphogluconate dehydrogenase (6PGD), provided strong evidence that the parent contributing the 2n gamete in both 3x cultivars was the female.
A DNA extraction protocol was developed for tissues from woody species. DNA was extracted successfully from 11 species and five different types of tissues and was suitable for RAPD and restriction analysis. Spermine precipitation was used to further purify DNA. The protocol can be used for large-scale analysis and mini-preparations.
Two random amplified polymorphic DNA (RAPD) markers linked to En, the gene conferring resistance to pea enation mosaic virus in pea, were identified and the DNA fragments were cloned and partially sequenced. Allele-specific associated primers for each cloned DNA fragment were developed and used in screening F2 populations. One marker, P256900, mapped very near Adh-1, about 6 cM from En. The other marker, B500400, was located about 8 cM from En on the same side as P256900.
Isozyme phenotypes were used to identify two (P2 and P3) of the six monosomic alien addition lines that have been isolated from a Cucurbita moschata × C. palmata hybrid. Phenotype P2 displayed the C. palmata fumarase isozyme, whereas P3 exhibited two glucose phosphate isomerases and an aspartate aminotransferase derived from C. palmata. P2 also possessed the hard rind trait characteristic of C. palmata. Both the biochemical and the morphological phenotypes were inherited in a non-Mendelian fashion, and no recombination was observed within either the P2 or P3 set of characters. It was concluded that the loci coding fumarase and hard rind were situated on the alien chromosome in P2 trisomics and that the other three loci were on a 2nd C. palmata chromosome possessed by the P3 line. The loci responsible for other C. palmata isozymes either were not expressed or were not located on any of the five C. palmata chromosomes represented in the alien addition lines.
The efficiency of marker-assisted selection for powdery mildew (Uncinula necator (Schw.) Burr) resistance in grapes (Vitis L. sp.) was studied using molecular markers associated with a major QTL (quantitative trait loci) for this trait. Initially, genetic maps were constructed from a segregating population of the cross `Horizon' × Illinois 547-1 (a hybrid between V. rupestris Scheele and V. cinerea Engelm.). A major QTL from Ill. 547-1, the resistant parent, explained 41% of the variation. One RAPD (randomly amplified polymorphic DNA) marker and one AFLP (amplified fragment length polymorphism) marker, obtained by bulked segregant analysis, showed the highest association with powdery mildew resistance in the mapping population. Segregation of the QTL was followed in different crosses by CAPS (cleaved amplified polymorphic sequence) markers developed from these two markers. An allele-specific amplified polymorphism that segregates as present/absent was also developed from the CS25b locus. Powdery mildew resistance was evaluated visually on a 1 to 5 scale in four different seedling populations. Two populations originated from crosses using Ill. 547-1 as the resistant parent. Two other populations were from crosses with NY88.0514.03, a resistant seedling from the original `Horizon' × Ill. 547-1 mapping population. Segregation ratio distortions were observed in some crosses. In these cases, the allele associated with the QTL for powdery mildew resistance was less frequent than the alternate allele. In all crosses, the markers were closely associated with resistance. If selection were based on markers, the percentage of susceptible individuals (classes 4 and 5) would decrease from 24% to 52% to 2% to 18%. Selection efficiency was greatest in crosses where segregation distortion was most intense.
DNA primers for 37 genes have been developed in pea (Pisum sativum L.). Two-thirds of these primers also amplify orthologous sequences in lentil (Lens culinaris). The primers were designed to be complementary to highly conserved sequences in exons of known genes. In addition, most of the priming sequences were selected to be 1000 to 3000 bp distant on the genomic DNA and to amplify a fragment that contained at least one intron. Segregating sequence polymorphism in mapping populations of recombinant inbred lines (RILs) derived from wide crosses in Pisum was observed by restriction of the amplified fragment with endonucleases recognizing four-base restriction sites. Successful mapping of 36 of these genes in pea demonstrated the utility of these primers for mapping, and it appears likely that the primers should have general utility for comparative mapping in legumes.